Genetically modified rodent with an inducible ACVR1 mutation in exon 7 that causes ectopic bone formation

Abstract

A genetically modified rodent is provided that comprises a modified Acvr1 gene that comprises a conditional altered exon 7 encoding R258G in anti sense orientation, flanked by site-specific recombinase recognition sites, wherein the altered exon is inverted to sense orientation upon action of a recombinase, resulting in ectopic bone formation.

What is claimed is: 
     
       1. A genetically modified rodent whose genome comprises a modified Activin A receptor type 1 (ACVR1) gene within an endogenous rodent ACVR1 locus, wherein the modified ACVR1 gene comprises:
 a) an ACVR1 exon 7 in sense orientation flanked by a first pair of site-specific recombinase recognition sites (SRRSs), wherein the ACVR1 exon 7 encodes the same amino acid sequence as a wild-type exon 7 of a human ACVR1 gene; and 
 b) a mutant exon 7 of a rodent ACVR1 gene in antisense orientation encoding a R258G substitution flanked by a second pair of SRRSs that are different from the first pair of SRRSs; 
 wherein the exon 7 in sense orientation:
 i) is the wild-type exon 7 of the human ACVR1 gene; or 
 ii) has decreased sequence identity to the mutant exon 7 of the rodent ACVR1 gene as compared to the wild-type exon 7 of the human ACVR1 gene; and 
 
 wherein the first and second pairs of SRRSs are oriented so that a recombinase can invert the mutant exon 7 into sense orientation, delete the exon 7 in sense orientation, and allow a mutant ACVR1 gene comprising the mutant exon 7 to be expressed resulting in ectopic bone formation. 
 
     
     
       2. The rodent of  claim 1 , wherein the exon 7 in sense orientation has decreased sequence identity to the mutant exon 7 of the rodent ACVR1 gene as compared to the wild-type exon 7 of the human ACVR1 gene. 
     
     
       3. The rodent of  claim 1 , wherein the genome of the rodent further comprises a nucleic acid sequence encoding the recombinase, and wherein the activity of the recombinase is inducible. 
     
     
       4. The rodent of  claim 3 , wherein the recombinase is Cre. 
     
     
       5. The rodent of  claim 4 , wherein the Cre is fused to a ligand binding domain of an estrogen receptor (ER) such that the activity of the Cre is induced by ligand binding to the ER. 
     
     
       6. The rodent of  claim 5 , wherein the ligand binding domain of the ER comprises T2 mutations. 
     
     
       7. The rodent of  claim 5 , wherein the ligand is tamoxifen. 
     
     
       8. The rodent of  claim 1 , wherein the genetically modified rodent is homozygous for the modified ACVR1 gene. 
     
     
       9. An offspring rodent derived from the rodent of  claim 1 , wherein the offspring rodent i) has a genome comprising the mutant ACVR1 gene comprising the mutant exon 7, and ii) expresses the mutant ACVR1 gene resulting in ectopic bone formation. 
     
     
       10. The rodent of  claim 1 , wherein the rodent is a mouse or rat. 
     
     
       11. An isolated genetically modified rodent cell or tissue whose genome comprises a modified Activin A receptor type 1 (ACVR1) gene within an endogenous rodent ACVR1 locus, wherein the modified ACVR1 gene comprises:
 a) an ACVR1 exon 7 in sense orientation flanked by a first pair of site-specific recombinase recognition sites (SRRSs), wherein the ACVR1 exon 7 encodes the same amino acid sequence as a wild-type exon 7 of a human ACVR1 gene; and 
 b) a mutant exon 7 of a rodent ACVR1 gene in antisense orientation encoding a R258G substitution flanked by a second pair of SRRSs that are different from the first pair of SRRSs; 
 wherein the exon 7 in sense orientation:
 i) is the wild-type exon 7 of the human ACVR1 gene; or 
 ii) has decreased sequence identity to the mutant exon 7 of the rodent ACVR1 gene as compared to the wild-type exon 7 of the human ACVR1 gene; and 
 
 wherein the first and second pairs of SRRSs are oriented so that a recombinase can invert the mutant exon 7 into sense orientation, delete the exon 7 in sense orientation, and allow a mutant ACVR1 gene comprising the mutant exon 7 to be expressed resulting in ectopic bone formation. 
 
     
     
       12. The isolated genetically modified rodent cell or tissue of  claim 11 , wherein the rodent is a mouse or a rat. 
     
     
       13. The isolated genetically modified rodent cell or tissue of  claim 11 , wherein the cell is an embryonic stem (ES) cell. 
     
     
       14. A method of making a genetically modified rodent whose genome comprises a modified Activin A receptor type 1 (ACVR1) gene, the method comprising:
 A) introducing a nucleic acid construct into an isolated rodent embryonic stem (ES) cell; 
 B) injecting the ES cell into a recipient rodent embryo; 
 C) implanting the embryo into a surrogate female such that a genetically modified rodent whose genome comprises a modified ACVR1 gene within an endogenous rodent ACVR1 locus is obtained, wherein the modified ACVR1 gene comprises:
 i) an ACVR1 exon 7 in sense orientation flanked by a first pair of site-specific recombinase recognition sites (SRRSs), wherein the ACVR1 exon 7 encodes the same amino acid sequence as a wild-type exon 7 of a human ACVR1 gene; and 
 ii) a mutant exon 7 of a rodent ACVR1 gene in antisense orientation encoding a R258G substitution flanked by a second pair of SRRSs from are different from the first pair of SRRSs; 
 
 wherein the exon 7 in sense orientation:
 a) is the wild-type exon 7 of the human ACVR1 gene; or 
 b) has decreased sequence identity to the mutant exon 7 of the rodent ACVR1 gene as compared to the wild-type exon 7 of the human ACVR1 gene; and 
 
 wherein the first and second pairs of SRRSs are oriented so that a recombinase can invert the mutant exon 7 into sense orientation, delete the exon 7 in sense orientation, and allow a mutant ACVR1 gene comprising the mutant exon 7 to be expressed resulting in ectopic bone formation. 
 
     
     
       15. The method of  claim 14 , wherein the rodent whose genome comprises the modified ACVR1 gene obtained is homozygous for the modified ACVR1 gene. 
     
     
       16. The method of  claim 14 , wherein the genome of the rodent ES cell comprises a nucleic acid sequence encoding the recombinase. 
     
     
       17. The method of  claim 14 , wherein the activity of the recombinase is inducible. 
     
     
       18. The method of  claim 17 , further comprising inducing the activity of the recombinase in a cell or tissue of the rodent whose genome comprises the modified ACVR1 gene such that the mutant exon 7 is inverted into sense orientation, the exon 7 in sense orientation is deleted, and the mutant ACVR1 gene comprising the mutant exon 7 is expressed and causes ectopic bone formation in the rodent. 
     
     
       19. The method of  claim 14 , wherein the recombinase is Cre. 
     
     
       20. The method of  claim 19 , wherein the Cre is fused to a ligand binding domain of an estrogen receptor (ER) such that the activity of the Cre is induced by ligand binding to the ER. 
     
     
       21. The method of  claim 20 , wherein the ligand binding domain of the ER comprises T2 mutations. 
     
     
       22. The method of  claim 20 , wherein the ligand is tamoxifen. 
     
     
       23. The method of  claim 14 , wherein the rodent is a mouse or rat.